11.1 Restriction and Modification Enzymes Genetic engineering: using in vitro techniques to alter genetic material in the laboratory Basic techniques include Restriction enzymes Gel electrophoresis Nucleic acid hybridization Nucleic acid probes Molecular cloning Cloning vectors © 2012 Pearson Education, Inc.

11.1 Restriction and Modification Enzymes Restriction enzymes: recognize specific DNA sequences and cut DNA at those sites Widespread among prokaryotes Rare in eukaryotes Protect prokaryotes from hostile foreign DNA (e.g., viral genomes) Essential for in vitro DNA manipulation © 2012 Pearson Education, Inc.

11.1 Restriction and Modification Enzymes Three classes of restriction enzymes Type II cleave DNA within their recognition sequence and are most useful for specific DNA manipulation (Figure 11.1a) Restriction enzymes recognize inverted repeat sequences (palindromes) Typically 4–8 base pairs long; EcoRI recognizes a 6-base-pair sequence Sticky ends or blunt ends © 2012 Pearson Education, Inc.

Single-stranded “sticky” ends Figure 11.1a Figure 11.1 Restriction and modification of DNA. Single-stranded “sticky” ends © 2012 Pearson Education, Inc.

11.1 Restriction and Modification Enzymes Restriction enzymes protect cell from invasion from foreign DNA Destroy foreign DNA Must protect their own DNA from inadvertent destruction © 2012 Pearson Education, Inc.

11.1 Restriction and Modification Enzymes Modification enzymes: protect cell’s DNA for restriction enzymes Chemically modify nucleotides in restriction recognition sequence Modification generally consists of methylation of DNA (Figure 11.1b) © 2012 Pearson Education, Inc.

Figure 11.1b Figure 11.1 Restriction and modification of DNA. © 2012 Pearson Education, Inc.

11.1 Restriction and Modification Enzymes Gel electrophoresis: separates DNA molecules based on size (Figure 11.2a) Electrophoresis uses an electrical field to separate charged molecules Gels are usually made of agarose, a polysaccharide Nucleic acids migrate through gel toward the positive electrode due to their negatively charged phosphate groups Gels can be stained with ethidium bromide and DNA can be visualized under UV light (Figure 11.2b) © 2012 Pearson Education, Inc.

Figure 11.2a Figure 11.2 Agarose gel electrophoresis of DNA. © 2012 Pearson Education, Inc.

 A B C D Size in base pairs Size in base pairs 5000 4000 3000 2000 Figure 11.2b  A B C D Size in base pairs Size in base pairs 5000 4000 3000 2000 1800 1000 Figure 11.2 Agarose gel electrophoresis of DNA. 500  © 2012 Pearson Education, Inc.

11.1 Restriction and Modification Enzymes The same DNA that has been cut with different restriction enzymes will have different banding patterns on an agarose gel Size of fragments can be determined by comparison to a standard Restriction map: a map of the location of restriction enzyme cuts on a segment of DNA (Figure 11.3) © 2012 Pearson Education, Inc.

11.2 Nucleic Acid Hybridization Nucleic acid hybridization: base pairing of single strands of DNA or RNA from two different sources to give a hybrid double helix Segment of single-stranded DNA that is used in hybridization and has a predetermined identity is called a nucleic acid probe Southern blot: a hybridization procedure where DNA is in the gel and probe is RNA or DNA Northern blot: RNA is in the gel © 2012 Pearson Education, Inc.

Figure 11.4 Figure 11.4 Southern blotting. © 2012 Pearson Education, Inc.

11.3 Essentials of Molecular Cloning Molecular cloning: isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated Three main steps of gene cloning (Figure 11.5): Isolation and fragmentation of source DNA Insertion of DNA fragment into cloning vector Introduction of cloned DNA into host organism © 2012 Pearson Education, Inc.

Introduction of recombinant vector into a host Figure 11.5 Foreign DNA Cut with restriction enzyme Add vector cut with same restriction enzyme Sticky ends Vector Add DNA ligase to form recombinant molecules Figure 11.5 Major steps in gene cloning. Cloned DNA Introduction of recombinant vector into a host © 2012 Pearson Education, Inc.

11.3 Essentials of Molecular Cloning Isolation and fragmentation of source DNA Source DNA can be genomic DNA, RNA, or PCR-amplified fragments Genomic DNA must first be restriction digested © 2012 Pearson Education, Inc.

11.3 Essentials of Molecular Cloning Insertion of DNA fragment into cloning vector Most vectors are derived from plasmids or viruses DNA is generally inserted in vitro DNA ligase: enzyme that joins two DNA molecules Works with sticky or blunt ends © 2012 Pearson Education, Inc.

11.3 Essentials of Molecular Cloning Introduction of cloned DNA into host organism Transformation is often used to get recombinant DNA into host Some cells will contain desired cloned gene, while other cells will have other cloned genes Gene library: mixture of cells containing a variety of genes Shotgun cloning: gene libraries made by cloning random genome fragments Animation: Recombinant DNA © 2012 Pearson Education, Inc.

11.3 Essentials of Molecular Cloning Essential to detect the correct clone Initial screen: antibiotic resistance, plaque formation Often sufficient for cloning of PCR-generated DNA sequences If working with a heterogeneous gene library you may need to look more closely © 2012 Pearson Education, Inc.

Transformant colonies growing on agar surface Figure 11.6 Transformant colonies growing on agar surface Replica-plate onto membrane filter Partially lyse cells; add specific antibody; add agent to detect bound antibody in radiolabeled form Lyse bacteria and denature DNA; add RNA or DNA probe (radioactive); wash out unbound radioactivity Figure 11.6 Finding the right clone. Autoradiograph to detect radioactivity X-ray film Positive colonies © 2012 Pearson Education, Inc.

11.4 Molecular Methods for Mutagenesis Synthetic DNA Systems are available for de novo synthesis of DNA Oligonucleotides of 100 bases can be made Multiple oligonucleotides can be ligated together Synthesized DNA is used for primers and probes, and in site-directed mutagenesis © 2012 Pearson Education, Inc.

11.4 Molecular Methods for Mutagenesis Conventional mutagens produce mutations at random Site-directed mutagenesis: performed in vitro and introduces mutations at a precise location (Figure 11.7) Can be used to assess the activity of specific amino acids in a protein Structural biologists have gained significant insight using this tool © 2012 Pearson Education, Inc.

single-stranded vector Source Figure 11.7 Clone into single-stranded vector Source Single-stranded DNA from M13 phage Add synthetic oligonucleotide with one base mismatch Base-pairing with source gene Extend single strand with DNA polymerase Figure 11.7 Site-directed mutagenesis using synthetic DNA. Transformation and selection Clone and select mutant © 2012 Pearson Education, Inc.

11.4 Molecular Methods for Mutagenesis Cassette mutagenesis and knockout mutations DNA fragment can be cut, excised, and replaced by a synthetic DNA fragment (DNA cassettes or cartridges) The process is known as cassette mutagenesis Gene disruption is when cassettes are inserted into the middle of the gene (Figure 11.8) Gene disruption causes knockout mutations © 2012 Pearson Education, Inc.

Gene X knockout Figure 11.8 EcoRI cut sites () Gene X Kanamycin cassette Cut with EcoRI and ligate BamHI cut site Cut with BamHI and transform into cell with wild-type gene X Linearized plasmid Sites of recombination Figure 11.8 Gene disruption by cassette mutagenesis. Chromosome Recombination and selection for kanamycin-resistant cells Gene X knockout © 2012 Pearson Education, Inc.

Figure 11.9 Figure 11.9 Green fluorescent protein (GFP). © 2012 Pearson Education, Inc.

Target gene Reporter gene Gene fusion Promoter Coding sequence Figure 11.10 Target gene Promoter Coding sequence Reporter gene Promoter Coding sequence Cut and ligate Gene fusion Promoter Reporter is expressed under control of target gene promoter Figure 11.10 Construction and use of gene fusions. Reporter enzyme Substrate Colored product © 2012 Pearson Education, Inc.

11.6 Plasmids as Cloning Vectors Plasmids are natural vectors and have useful properties as cloning vectors Small size; easy to isolate DNA Independent origin of replication Multiple copy number; get multiple copies of cloned gene per cell Presence of selectable markers Vector transfer carried out by chemical transformation or electroporation © 2012 Pearson Education, Inc.

11.6 Plasmids as Cloning Vectors pUC19 is a common cloning vector (Figure 11.11) Modified ColE1 plasmid Contains ampicillin resistance and lacZ genes Contains polylinker (multiple cloning site) within lacZ gene © 2012 Pearson Education, Inc.

Origin of DNA replication Figure 11.11 Order of restriction enzyme cut sites in polylinker Ampicillin resistance lacZ ApoI - EcoRI BanII - SacI Acc651 - KpnI AvaI - BsoBI - SmaI - XmaI BamHI XbaI AccI - HincII - SalI BspMI - BfuAI SbfI PstI SphI HindIII Polylinker pUC19 2686 base pairs Figure 11.11 Cloning vector plasmid pUC19. lacI Origin of DNA replication © 2012 Pearson Education, Inc.

11.6 Plasmids as Cloning Vectors Blue/white screening Blue colonies do not have vector with foreign DNA inserted White colonies have foreign DNA inserted Insertional inactivation: lacZ gene is inactivated by insertion of foreign DNA (Figure 11.12) Inactivated lacZ cannot process Xgal; blue color does not develop © 2012 Pearson Education, Inc.

Vector plus foreign DNA insert Figure 11.12 lacZ AmpR Foreign DNA Vector Digestion with restriction enzyme Join with DNA ligase Opened vector Figure 11.12 Cloning into the plasmid vector pUC19. Recyclized vector without insert Vector plus foreign DNA insert Transform into Escherichia coli and select on ampicillin plates containing Xgal Transformants blue (-galactosidase active) Transformants white (-galactosidase inactive) © 2012 Pearson Education, Inc.

11.7 Hosts for Cloning Vectors Ideal hosts should be Capable of rapid growth in inexpensive medium Nonpathogenic Capable of incorporating DNA Genetically stable in culture Equipped with appropriate enzymes to allow replication of the vector Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae © 2012 Pearson Education, Inc.

Bacteria Eukaryote Escherichia coli Bacillus subtilis Figure 11.13 Bacteria Eukaryote Escherichia coli Bacillus subtilis Saccharomyces cerevisiae Well-developed genetics Many strains available Best known bacterium Easily transformed Nonpathogenic Naturally secretes proteins Endospore formation simplifies culture Well-developed genetics Nonpathogenic Can process mRNA and proteins Easy to grow Figure 11.13 Hosts for molecular cloning. Potentially pathogenic Periplasm traps proteins Genetically unstable Genetics less developed than in E. coli Plasmids unstable Will not replicate most bacterial plasmids Advantages Disadvantages © 2012 Pearson Education, Inc.

11.5 Gene Fusions and Reporter Genes Encode proteins that are easy to detect and assay (Figure 11.9) Examples: lacZ, luciferase, GFP genes Gene fusions Promoters or coding sequences of genes of interest can be swapped with those of reporter genes to elucidate gene regulation under various conditions (Figure 11.10) © 2012 Pearson Education, Inc.

11.8 Shuttle Vectors and Expression Vectors Expression vectors: allow experimenter to control the expression of cloned genes (Figure 11.16) Based on transcriptional control Allow for high levels of protein expression Strong promoters lac, trp, tac, trc, lambda PL Effective transcription terminators are used to prevent expression of other genes on the plasmid © 2012 Pearson Education, Inc.

Polylinker (cloning site) Figure 11.16 trc promoter lacO S/D lacI Polylinker (cloning site) T1 T2 Figure 11.16 Genetic map of the expression vector pSE420. Ampicillin resistance Origin of DNA replication © 2012 Pearson Education, Inc.

11.8 Shuttle Vectors and Expression Vectors In T7 expression vectors, cloned genes are placed under control of the T7 promoter (Figure 11.17) Gene for T7 RNA polymerase present and under control of easily regulated system (e.g., lac) T7 RNA polymerase recognizes only T7 promoters Transcribes only cloned genes Shuts down host transcription © 2012 Pearson Education, Inc.

Gene for T7 RNA polymerase Figure 11.17 Induce lac promoter with IPTG T7 RNA polymerase Gene product lac operator Gene for T7 RNA polymerase lac promoter T7 promoter Cloned gene pET plasmid Figure 11.17 The T7 expression system. Chromosome lacl © 2012 Pearson Education, Inc.

11.8 Shuttle Vectors and Expression Vectors mRNA produced must be efficiently translated and there are problems with this always happening Bacterial ribosome binding sites are not present in eukaryotic genomes Differences in codon usage between organisms Eukaryotic genes containing introns will not be expressed properly in prokaryotes © 2012 Pearson Education, Inc.